Peroxidase Activity in Poplar Inoculated with Compatible and Incompetent Isolates of Paxillus involutus
HAYATI Journal of Biosciences, June 2007, p 49-53
ISSN: 1978-3019
Vol. 14, No. 2
Peroxidase Activity in Poplar Inoculated with
Compatible and Incompetent Isolates of Paxillus involutus
ABDUL GAFUR1∗∗, ANDRES SCHUTZENDUBEL2, ANDREA POLLE2
1
FiberOne Research and Development, PT Riau Andalan Pulp and Paper, Town Site I, Pangkalan Kerinci 28300, Indonesia
2
Institute for Forest Botany, University of Göttingen, Büsgenweg 2, D-37077 Göttingen, Germany
Received April 25, 2006/Accepted May 14, 2007
Peroxidase activity of the hybrid poplar Populus x canescens (Ait.) Sm. (= P. tremula L. x P. alba L.) inoculated with
compatible and incompetent isolates of Paxillus involutus (Batsch) Fr. was investigated. Screening of the ectomycorrhizal
fungal isolates was initiated with exploration of mycelial growth characteristics and mycorrhizal ability in vitro with
poplar. Both traits varied within the fungus although they did not seem to be genetically correlated. While isolates SCO1, NAU,
and 031 grew faster than others, only isolates MAJ, SCO1, and 031 were able to form ectomycorrhiza with poplar. Isolates
MAJ (compatible) and NAU (incompetent) were subsequently selected for further experiments. Activity of peroxidase, one of the
defense-related enzymes, was examined in pure culture and short root components of compatible and incompetent interactions
between poplar and P. involutus. Peroxidase activities increased significantly in poplar inoculated with incompetent
isolate of the fungus compared to control, while induction of the same enzyme was not detected in compatible associations.
Key words: ectomycorrhiza, Paxillus involutus, peroxidase, plant defense, poplar
___________________________________________________________________________
INTRODUCTION
Despite widespread recognition of important roles of
ectomycorrhizal fungi, a little is known about their
physiological and molecular interactions with plants. The
nature of these mutually symbiotic associations between fungi
and roots is still in its infancy and remains poorly understood.
There have been indications, however, that compared with
much tighter plant-pathogen interactions, mycorrhizal
symbiosis lacks specificity between the fungi and the plants.
It is clear that we need information in a number of areas
including the genes and proteins present in symbiotic tissues
and how they are regulated. The question of whether plants
recognize the presence of symbiont at all, or rather that the
defense responses are repressed by the symbiotic fungus in
order to allow a compatible interaction is yet to be elucidated.
In plant-mycorrhizal associations, a number of different
approaches to investigate mechanisms occurs during the
symbiosis have been under taken. Most of the strategies,
however, have involved comparisons with other plant-microbe
interactions about which we have more extensive information
(Smith 1999). This includes production of hydrogen peroxide
(H2O2) and peroxidases (Morandi et al. 1984; GianinazziPearson et al. 1992; Olson & Varner 1993; Lamb & Dixon
1997; Karpinski et al. 1999; Pellinen et al. 1999; OrozcoCárdenas et al. 2001; Salzer & Boller 2001; Schützendübel et
al. 2001; Tagu et al. 2001; Gafur et al. 2004) and the expression
of genes coding for pathogenesis-related (PR) protein such
as chitinases (Gianinazzi 1991; Dumas-Gaudot et al. 1992;
Gianinazzi-Pearson et al. 1992). Information on similarities and
differences between the interactions will subsequently lead
to a good understanding of the molecular nature of mycorrhizal
_________________
∗
Corresponding author. Phone/Fax: +62-761-95550 ext 5222,
Fax: +62-761-95306, E-mail: [email protected]
development which can be linked to the physiological and
biochemical function of mycorrhizal symbioses of different
types (Smith 1999).
The present study was aimed at investigating whether
active defense mechanisms, such as activation of peroxidases,
are induced in hybrid poplar Populus x canescens (Ait.) Sm.
(= P. tremula L. x P. alba L.) in response to inoculation with
Paxillus involutus (Batsch) Fr. (Basidiomycetes, Boletales,
Paxillaceae). The involvement of peroxidases in induction of
plant resistance has been confirmed. Generally, peroxidases
enhance their activity after pathogen attack (Lebeda et al.
2001). To facilitate this exploration, in vitro, rapid synthesis
of ectomycorrhiza under aseptic conditions and in systems
which produce uniform infection needed to be established
(Gafur et al. 2005). Isolates of P. involutus with different
mycorrhizal abilities were also required for perfect comparisons
of the interactions. As the fungus represents broad-range
host fungal species (Langenfeld-Heyser et al. 2007), the
poplar-P. involutus system described in this study may also
be employed in exploration of other ectomycorrhizal
interactions.
MATERIALS AND METHODS
Plant Culture, Fungal Culture, and Growth Media. After
cutting, plantlets of hybrid poplar were grown for three to
four weeks on MS medium (Murashige & Skoog 1962) to allow
sufficient rooting under sterile conditions before use for
ectomycorrhization. To ensure optimal growing conditions
for the plant, special attention was focused to the humidity of
the growing system, as young poplars are sensitive to low
humidity. Eleven isolates of P. involutus used in this study
(Table 1) were stock cultures available at the Institute for
Forest Botany, University of Göttingen, Göttingen, Germany.
50
GAFUR ET AL.
They were collected from various hosts and environmental
conditions in different places of Germany and other European
countries. Prior to use as inoculum for the aseptic synthesis
of ectomycorrhiza, five small plugs (10 mm diameter) of actively
growing mycelia of P. involutus were transferred onto Modified
Melin Norkrans (MMN) agar medium with further
modifications and covered with cellophane. The medium
contains (l -1) 0.5 g KH 2PO 4, 0.25 g (NH 4)2SO 4 , 0.15 g
MgSO4·7H20, 1 ml of 1% FeCl3, 0.05 g CaCl2, 0.025 g NaCl, 5 g
glucose, 3 g malt extract, 100 µg thiamine HCl. After
addition of 2.5 ml trace elements, the pH was adjusted to
4.5. In order for the agar not to dry out, 30-35 ml of media
was added to each of the 9 cm Petri dish.
Ectomycorrhizal Ability and Growth Characteristics of
Fungal Isolates. For physiological investigations, both
compatible and incompetent isolates that express optimal
growing ability in pure culture were required. All isolates
of P. involutus used in the present study were tested both for
their capability to form ectomycorrhizal associations with
poplar and for growth characteristics. Ectomycorrhization was
carried out according to the technique developed by Hampp
et al. (1996) with modifications. Rooted plantlets from sterile
culture (about three weeks old) were carefully freed from agar
particles and, for better handling, the lowermost leaves were
removed. The root system of the plantlets was transferred
directly to Petri dishes (9 cm diameter) containing 30-35 ml of
modified MMN agar covered with a sheet of cellophane on
which five plugs of fungal inoculum had been pregrown for
one week. The root was carefully expanded to allow optimal
contact with the agar surface. The lower end of the shoot was
positioned in an opening of the sidewall of the dish punctured
with a hot needle serving as an exit gate of the plants from the
Petri dish. The Petri dish was closed, sealed with a strip of
parafilm and then wrapped with aluminum foil to keep the
roots in darkness. Symbiont culture was maintained in a special
planting chamber, the bottom of which was covered with wet
filter papers. As additional measures to prevent evaporation,
the planting chamber was covered with a plastic cover. Quick
transfer and high humidity were essential for keeping the
plantlets alive. The whole system was maintained at 20 oC
under continuous illumination. Roots were examined for
mycorrhizal formation after one, four, and seven weeks of
inoculation. Control plants were grown under the absence of
other organisms. In the meantime, fungal growth
characteristics were expressed as mycelial radial growth
measured three weeks after incubation. Six plates of each
isolate were examined. The cultures were maintained in Petri
dishes by successive transfer on modified MMN agar medium.
Histochemical Staining for Mantle and Hartig Net
Localization. Root tips of control, non-mycorrhizal and
mycorrhizal tissues were vacuum infiltrated with freshly
prepared 5 mM CeCl 3 in 50 mM 3-(N-morpholino)propanesulfonic acid at pH 7.2 for 30 min. Tissues were then
fixed in 1.25% (v/v) glutaraldehyde and 1.25% (v/v)
paraformaldehyde in 50 mM sodium cacodylate buffer (CAB),
pH 7.2, for 1 hr at room temperature and kept overnight at 4 oC.
After fixation, tissues were washed twice for 10 min in CAB
and postfixed for 45 min in 1% (v/v) osmium tetroxide in CAB
HAYATI J Biosci
and dehydrated in a graded acetone series (30, 50, 70, 80, 90,
and 100% [v/v]). The tissues were then progressively
embedded in rising concentrations of acetone-resin mixtures,
and finally incubated in two 24-hr changes of pure epoxy
resin before polymerization at 60 oC for 48 hr. Sections of
control, compatible, and incompetent root tissues were
examined under microscope and subsequently photographed
with a digital camera (Coolpix 990, Nikon, Tokyo) for
observation of anatomical characteristics of the interactions
with the emphasis on the formation of mantle and Hartig net.
Peroxidase Activity. Peroxidase accumulation induced as
plant defense responses was investigated in the present
poplar-Paxillus interactions. The enzymatic activities of the
guaiacol peroxidase were determined according to the method
of Pütter (1970), modified after Polle et al. (1990). Briefly, the
assay reaction contained 500 µl of 100 mM potassium
phosphate buffer pH 5.25, 400 µl of 100 mM guaiacol, 50 µl
extract supernatant, and 50 µl of 200 mM H2O2. Assays were
initiated by addition of H2O2 and the change of optical density
at 436 nm was measured for 5 min at 25 oC (Beckman DU 640,
Beckman Instruments Inc., CA, USA). Peroxidase activity was
expressed as the change in optical density on the basis of
protein contents. Short roots of control, compatible, and
incompetent associations were harvested after one, four, and
seven weeks, respectively, of inoculation for root sample
preparations. Five plants were examined for each host-fungal
isolate interaction and incubation period combination.
Collected roots were powdered in liquid nitrogen using mortar
and pestle. The powder was extracted 15 min at 4 oC in 1 ml of
cold extraction buffer (10 mM Tris-HCl, 10 mM NaHCO3,
10 mM MgCl2, 0.1 mM Na-EDTA, 10 mM Mercaptoethanol,
1 mM PMSF, 2% polyvinylpolypyrrolidone (PVPP), 0.1% [v/v]
Triton X-100). The extract was centrifuged (30 min, 24,000
x g, 4 oC) and the supernatant was passed through a Sephadex
G-25 column (NAP-5 column, Amersham Pharmacia Biotech
AB, Uppsala, Sweden) and equilibrated with 100 mM
potassium phosphate, pH 7.8. For fungal pure culture
extraction, mycelia were grown for three weeks in Petri dishes
on modified MMN agar. Suspension cultures were derived
by homogenization of fungal inoculum from these cultures in
liquid modified MMN medium and grown for two weeks on a
shaker. After another homogenization, the suspension was
transferred to new liquid medium for physiological studies.
After incubation for five and ten days, respectively, fungal
mycelia were homogenized in a frozen state. Five flasks were
examined for each treatment. Extraction procedure was as
explained above for plant roots with only minor modifications.
RESULTS
Selection of Fungal Isolates. Three (MAJ, SCO1, and 031)
of the 11 isolates of P. involutus tested were found to
consistently form ectomycorrhiza with poplar, whereas the
others failed (Table 1). Failure to associate successfully may
indicate that the isolates were incompetent with poplar. The
table also showed variations in the growth ability within the
species. All fungal isolates grew on modified MMN agar,
except isolate B12. However, the growth form of mycelia varied
Vol. 14, 2007
POPLAR-PAXILLUS INVOLUTUS PHYSIOLOGICAL INTERACTION 51
Table 1. Origin, host, mycelial radial growth, and ectomycorrhizal
ability with hybrid poplar of isolates of P. involutus (Batsch)
Fr. used in this study
Isolate Origin
Host* Radial growth (mm)** Ectomycorrhiza***
43.00 ± 4.16
B05 Belgium ND
46.83 ± 5.58
B08 Belgium ND
00.00 ± 0.00
B12 Belgium ND
B13 Belgium ND
27.17 ± 1.57
49.50 ± 2.22
+
MAJ France
Poplar
Oak
78.83 ± 2.48
NAU France
Birch/pine
31.00 ± 2.38
P 1 9 Poland
+
85.00 ± 0.00
SCO1 Scotland ND
65.17 ± 3.08
+
031 Germany Spruce
27.83 ± 0.69
032 Germany Spruce
26.17 ± 1.57
533 Germany Spruce
*ND: not determined; **Radial growth was measured three weeks
after the fungi were grown on MMN. Values are expressed as means of
six replicates; ***Ectomycorrhiza was formed (+) or not (-)
Control
among isolates, with particular isolates (SCO1, NAU, and 031)
producing more rapid and dense mycelia during growth on
agar-based media than others. Two isolates with reasonable
growth characteristics, compatible MAJ, and incompetent
NAU (Figure 1), were selected for further experiments. MAJ
was selected for the compatible interaction because the isolate
produced the most consistent ectomycorrhizal association
with poplar.
Morphological and Anatomical Characteristics. Root tips
morphological changes of compatible association
characteristics of mycorrhizal formation were visible one week
after inoculation. The mycorrhiza continued to develop and
they were mature by five to seven weeks. Development of
poplar both grown in the absence and presence of P. involutus
under currently described Petri dish system occurred normally.
Incompetent
Compatible
Figure 1. Root systems of poplar in control plant (left) and plants inoculated with compatible MAJ (middle) and incompetent NAU (right)
P. involutus isolates.
Control
Compatible
5.0 mm
Incompetent
2.5 mm
2.5 mm
R
M
H
0.1 mm
Figure 2. Morphology of poplar root tips (top) and anatomical characteristics of the cross sections of the root tips (bottom) in control,
compatible, and incompeten isolates of P. involutus four weeks after inoculation. H: Hartig net, M: mantle, R: root hair. Control plants
were grown in the absence of the fungus.
52
GAFUR ET AL.
HAYATI J Biosci
600
nkat/g protein
500
400
300
200
100
0
R_COMP
R_NONCOMP
R_CTRL
Figure 3. Peroxidase activities in interaction between poplar and P.
involutus. Data were taken four (left bar) and seven (right
bar) weeks after inoculation. Values are expressed as means
of five replicates. Peroxidase activities were induced only
in incompetent interactions (R_NONCOMP), not in
compatible associations (R_COMP) or in control plants
(R_CTRL).
All the plants exhibited a well-developed root system (Figure
1). Mycorrhiza formed by association between poplar and
compatible isolate of P. involutus were initially pale white to
yellowish and then became brown with age. Mycorrhized
plants in this study were characterized among others by
dramatic changes in root morphology such as stimulation of
lateral root production and suppression of root hair formation.
As shown in Figure 2 (top), root hair formation was restricted
in compatible association. Anatomical characteristics of the
roots of control, compatible, and incompetent associations
were shown in Figure 2 (bottom). The mantle of compatible
isolate surface consisted of loose web-like prosenchymatous
elements. Mantle thickness varied and was irregularly layered.
Hartig net developed showed intercellular penetration by
hyphae, mostly in the form of a single hyphal row, to the first
cortical layer. Upon maturity, the Hartig net fully enveloped
the cells of epidermal layer forming a periepidermal net.
Peroxidase Accumulation. Peroxidase activity increased
significantly in roots of poplar in response to incompetent
isolate of P. involutus (Figure 3). Elevated levels of the
enzymatic activity were maintained seven weeks after
inoculation. In contrast, no significant changes in peroxidase
activity were observed in compatible tissues after inoculation
with mycorrhizal isolates MAJ or in control roots.
DISCUSSION
The results of the present study support the view that
P. involutus has a broad variation of host range at the isolate
level, as this has been previously reported by Heslin and
Douglas (1986) and Molina and Trappe (1982). A number of
studies also attest to significant variation in the ability of
isolates of other fungal species to form ectomycorrhiza with
particular host taxa (Bonfante et al. 1998; Cairney 1999). Similar
growth characteristics of different ectomycorrhizal fungal
species have also been earlier reported by KieliszewskaRokicka (1992) and Littke et al. (1984).
Despite the fact that both growth characteristics and
mycorrhizal ability of P. involutus vary among the tested
isolates, the two traits do not seem to be correlated in the
fungus. For example, isolates NAU and 031 whose growth
rate is at a similar level, differ in their ability to form
ectomycorrhiza. While isolate 031 was able to form
ectomycorrhiza with poplar, isolate NAU failed. In contrast,
the growth rate of compatible isolates MAJ and 031 was
different. Nevertheless, this lack of correlation between the
two traits of mycelial growth and ectomycorrhizal ability
enabled us to select isolates perfectly suitable for further
experiments, i.e. compatible and incompetent isolates with
optimal growth ability.
Increased peroxidase activity has been reported in many
pathogenic interactions between plants and fungi (Svalheim
& Robertsen 1990; Asiegbu et al. 1994; Fossdal et al. 2001).
The fact that overall peroxidase activity was not significantly
increased in the mycorrhizal roots of poplar supports earlier
observations that mycorrhizal colonization does not activate
full-scale plant-defense responses (Nylund 1981; Nylund 1987;
Münzenberger et al. 1997) or elicitation of defense responses
is avoided (Mohr et al. 1998). In the meantime, failure to detect
peroxidase activities to an appreciable amount in short roots
of control plants may well explain that defense-related
peroxidases, in the absence of root microbial pathogens or
other organisms, are not expressed.
As a defense mechanism, plants are able to respond to
invading organisms in a variety of ways including production
of components which modify cell walls including peroxidases,
making them more difficult to penetrate (Hahlbrock et al. 1986).
In symbiotic associations, on the other hand, both cell wall
bound peroxidase and chitinase increased above control levels
during the first days of mycorrhization, only to return to or
fall below those of non-mycorrhizal roots at later stages of
established symbiosis (Salzer & Boller 2001). However,
Bonfante-Fasolo and Spanu (1992) pointed out that most of
the experiments had not always been of a comparative nature,
and a more systematic approach considering both compatible
and incompetent mycorrhizal and pathogenic associations
was needed. The present study, therefore, sheds some light
on and contributes to better understanding of the mechanism
of recognition in plant-microbe interactions.
Accumulation of peroxidase was not induced by all isolates
of P. involutus tested in pure culture (data not shown). All
isolates, including poplar-incompetent, did not seem to induce
peroxidase accumulation to an appreciable amount. Similar
results that no peroxidase activities are detected in extracts of
extramatrical mycelium or pure culture mycelium have also
been reported in different isolates of Suillus bovinus and
P. involutus by Timonen and Sen (1998). Cairney and Burke
(1998) also noted that there is currently no convincing evidence
to support production of peroxidase activity by
ectomycorrhizal fungi in vitro.
Isolates of MAJ and NAU served as perfect models to
investigate physiological interactions between poplar and
P. involutus. At the genomic level we have shown that the
two isolates are very similar with DNA sequence identity of
98.9% although the difference is more significant in terms of
expressed genes after contact with birch roots (Le Quéré et
al. 2004). Thus, the currently described novel system should
POPLAR-PAXILLUS INVOLUTUS PHYSIOLOGICAL INTERACTION 53
Vol. 14, 2007
also enable investigations on ectomycorrhizal interactions in
different systems.
ACKNOWLEDGEMENT
This work was partially supported by a research fellowship
from the Alexander von Humboldt Foundation, Bonn, Germany
to Abdul Gafur. We thank Rosemarie Langenfeld-Heyser,
Christine Kettner, and Gisbert Langer-Kettner for technical
assistance.
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ISSN: 1978-3019
Vol. 14, No. 2
Peroxidase Activity in Poplar Inoculated with
Compatible and Incompetent Isolates of Paxillus involutus
ABDUL GAFUR1∗∗, ANDRES SCHUTZENDUBEL2, ANDREA POLLE2
1
FiberOne Research and Development, PT Riau Andalan Pulp and Paper, Town Site I, Pangkalan Kerinci 28300, Indonesia
2
Institute for Forest Botany, University of Göttingen, Büsgenweg 2, D-37077 Göttingen, Germany
Received April 25, 2006/Accepted May 14, 2007
Peroxidase activity of the hybrid poplar Populus x canescens (Ait.) Sm. (= P. tremula L. x P. alba L.) inoculated with
compatible and incompetent isolates of Paxillus involutus (Batsch) Fr. was investigated. Screening of the ectomycorrhizal
fungal isolates was initiated with exploration of mycelial growth characteristics and mycorrhizal ability in vitro with
poplar. Both traits varied within the fungus although they did not seem to be genetically correlated. While isolates SCO1, NAU,
and 031 grew faster than others, only isolates MAJ, SCO1, and 031 were able to form ectomycorrhiza with poplar. Isolates
MAJ (compatible) and NAU (incompetent) were subsequently selected for further experiments. Activity of peroxidase, one of the
defense-related enzymes, was examined in pure culture and short root components of compatible and incompetent interactions
between poplar and P. involutus. Peroxidase activities increased significantly in poplar inoculated with incompetent
isolate of the fungus compared to control, while induction of the same enzyme was not detected in compatible associations.
Key words: ectomycorrhiza, Paxillus involutus, peroxidase, plant defense, poplar
___________________________________________________________________________
INTRODUCTION
Despite widespread recognition of important roles of
ectomycorrhizal fungi, a little is known about their
physiological and molecular interactions with plants. The
nature of these mutually symbiotic associations between fungi
and roots is still in its infancy and remains poorly understood.
There have been indications, however, that compared with
much tighter plant-pathogen interactions, mycorrhizal
symbiosis lacks specificity between the fungi and the plants.
It is clear that we need information in a number of areas
including the genes and proteins present in symbiotic tissues
and how they are regulated. The question of whether plants
recognize the presence of symbiont at all, or rather that the
defense responses are repressed by the symbiotic fungus in
order to allow a compatible interaction is yet to be elucidated.
In plant-mycorrhizal associations, a number of different
approaches to investigate mechanisms occurs during the
symbiosis have been under taken. Most of the strategies,
however, have involved comparisons with other plant-microbe
interactions about which we have more extensive information
(Smith 1999). This includes production of hydrogen peroxide
(H2O2) and peroxidases (Morandi et al. 1984; GianinazziPearson et al. 1992; Olson & Varner 1993; Lamb & Dixon
1997; Karpinski et al. 1999; Pellinen et al. 1999; OrozcoCárdenas et al. 2001; Salzer & Boller 2001; Schützendübel et
al. 2001; Tagu et al. 2001; Gafur et al. 2004) and the expression
of genes coding for pathogenesis-related (PR) protein such
as chitinases (Gianinazzi 1991; Dumas-Gaudot et al. 1992;
Gianinazzi-Pearson et al. 1992). Information on similarities and
differences between the interactions will subsequently lead
to a good understanding of the molecular nature of mycorrhizal
_________________
∗
Corresponding author. Phone/Fax: +62-761-95550 ext 5222,
Fax: +62-761-95306, E-mail: [email protected]
development which can be linked to the physiological and
biochemical function of mycorrhizal symbioses of different
types (Smith 1999).
The present study was aimed at investigating whether
active defense mechanisms, such as activation of peroxidases,
are induced in hybrid poplar Populus x canescens (Ait.) Sm.
(= P. tremula L. x P. alba L.) in response to inoculation with
Paxillus involutus (Batsch) Fr. (Basidiomycetes, Boletales,
Paxillaceae). The involvement of peroxidases in induction of
plant resistance has been confirmed. Generally, peroxidases
enhance their activity after pathogen attack (Lebeda et al.
2001). To facilitate this exploration, in vitro, rapid synthesis
of ectomycorrhiza under aseptic conditions and in systems
which produce uniform infection needed to be established
(Gafur et al. 2005). Isolates of P. involutus with different
mycorrhizal abilities were also required for perfect comparisons
of the interactions. As the fungus represents broad-range
host fungal species (Langenfeld-Heyser et al. 2007), the
poplar-P. involutus system described in this study may also
be employed in exploration of other ectomycorrhizal
interactions.
MATERIALS AND METHODS
Plant Culture, Fungal Culture, and Growth Media. After
cutting, plantlets of hybrid poplar were grown for three to
four weeks on MS medium (Murashige & Skoog 1962) to allow
sufficient rooting under sterile conditions before use for
ectomycorrhization. To ensure optimal growing conditions
for the plant, special attention was focused to the humidity of
the growing system, as young poplars are sensitive to low
humidity. Eleven isolates of P. involutus used in this study
(Table 1) were stock cultures available at the Institute for
Forest Botany, University of Göttingen, Göttingen, Germany.
50
GAFUR ET AL.
They were collected from various hosts and environmental
conditions in different places of Germany and other European
countries. Prior to use as inoculum for the aseptic synthesis
of ectomycorrhiza, five small plugs (10 mm diameter) of actively
growing mycelia of P. involutus were transferred onto Modified
Melin Norkrans (MMN) agar medium with further
modifications and covered with cellophane. The medium
contains (l -1) 0.5 g KH 2PO 4, 0.25 g (NH 4)2SO 4 , 0.15 g
MgSO4·7H20, 1 ml of 1% FeCl3, 0.05 g CaCl2, 0.025 g NaCl, 5 g
glucose, 3 g malt extract, 100 µg thiamine HCl. After
addition of 2.5 ml trace elements, the pH was adjusted to
4.5. In order for the agar not to dry out, 30-35 ml of media
was added to each of the 9 cm Petri dish.
Ectomycorrhizal Ability and Growth Characteristics of
Fungal Isolates. For physiological investigations, both
compatible and incompetent isolates that express optimal
growing ability in pure culture were required. All isolates
of P. involutus used in the present study were tested both for
their capability to form ectomycorrhizal associations with
poplar and for growth characteristics. Ectomycorrhization was
carried out according to the technique developed by Hampp
et al. (1996) with modifications. Rooted plantlets from sterile
culture (about three weeks old) were carefully freed from agar
particles and, for better handling, the lowermost leaves were
removed. The root system of the plantlets was transferred
directly to Petri dishes (9 cm diameter) containing 30-35 ml of
modified MMN agar covered with a sheet of cellophane on
which five plugs of fungal inoculum had been pregrown for
one week. The root was carefully expanded to allow optimal
contact with the agar surface. The lower end of the shoot was
positioned in an opening of the sidewall of the dish punctured
with a hot needle serving as an exit gate of the plants from the
Petri dish. The Petri dish was closed, sealed with a strip of
parafilm and then wrapped with aluminum foil to keep the
roots in darkness. Symbiont culture was maintained in a special
planting chamber, the bottom of which was covered with wet
filter papers. As additional measures to prevent evaporation,
the planting chamber was covered with a plastic cover. Quick
transfer and high humidity were essential for keeping the
plantlets alive. The whole system was maintained at 20 oC
under continuous illumination. Roots were examined for
mycorrhizal formation after one, four, and seven weeks of
inoculation. Control plants were grown under the absence of
other organisms. In the meantime, fungal growth
characteristics were expressed as mycelial radial growth
measured three weeks after incubation. Six plates of each
isolate were examined. The cultures were maintained in Petri
dishes by successive transfer on modified MMN agar medium.
Histochemical Staining for Mantle and Hartig Net
Localization. Root tips of control, non-mycorrhizal and
mycorrhizal tissues were vacuum infiltrated with freshly
prepared 5 mM CeCl 3 in 50 mM 3-(N-morpholino)propanesulfonic acid at pH 7.2 for 30 min. Tissues were then
fixed in 1.25% (v/v) glutaraldehyde and 1.25% (v/v)
paraformaldehyde in 50 mM sodium cacodylate buffer (CAB),
pH 7.2, for 1 hr at room temperature and kept overnight at 4 oC.
After fixation, tissues were washed twice for 10 min in CAB
and postfixed for 45 min in 1% (v/v) osmium tetroxide in CAB
HAYATI J Biosci
and dehydrated in a graded acetone series (30, 50, 70, 80, 90,
and 100% [v/v]). The tissues were then progressively
embedded in rising concentrations of acetone-resin mixtures,
and finally incubated in two 24-hr changes of pure epoxy
resin before polymerization at 60 oC for 48 hr. Sections of
control, compatible, and incompetent root tissues were
examined under microscope and subsequently photographed
with a digital camera (Coolpix 990, Nikon, Tokyo) for
observation of anatomical characteristics of the interactions
with the emphasis on the formation of mantle and Hartig net.
Peroxidase Activity. Peroxidase accumulation induced as
plant defense responses was investigated in the present
poplar-Paxillus interactions. The enzymatic activities of the
guaiacol peroxidase were determined according to the method
of Pütter (1970), modified after Polle et al. (1990). Briefly, the
assay reaction contained 500 µl of 100 mM potassium
phosphate buffer pH 5.25, 400 µl of 100 mM guaiacol, 50 µl
extract supernatant, and 50 µl of 200 mM H2O2. Assays were
initiated by addition of H2O2 and the change of optical density
at 436 nm was measured for 5 min at 25 oC (Beckman DU 640,
Beckman Instruments Inc., CA, USA). Peroxidase activity was
expressed as the change in optical density on the basis of
protein contents. Short roots of control, compatible, and
incompetent associations were harvested after one, four, and
seven weeks, respectively, of inoculation for root sample
preparations. Five plants were examined for each host-fungal
isolate interaction and incubation period combination.
Collected roots were powdered in liquid nitrogen using mortar
and pestle. The powder was extracted 15 min at 4 oC in 1 ml of
cold extraction buffer (10 mM Tris-HCl, 10 mM NaHCO3,
10 mM MgCl2, 0.1 mM Na-EDTA, 10 mM Mercaptoethanol,
1 mM PMSF, 2% polyvinylpolypyrrolidone (PVPP), 0.1% [v/v]
Triton X-100). The extract was centrifuged (30 min, 24,000
x g, 4 oC) and the supernatant was passed through a Sephadex
G-25 column (NAP-5 column, Amersham Pharmacia Biotech
AB, Uppsala, Sweden) and equilibrated with 100 mM
potassium phosphate, pH 7.8. For fungal pure culture
extraction, mycelia were grown for three weeks in Petri dishes
on modified MMN agar. Suspension cultures were derived
by homogenization of fungal inoculum from these cultures in
liquid modified MMN medium and grown for two weeks on a
shaker. After another homogenization, the suspension was
transferred to new liquid medium for physiological studies.
After incubation for five and ten days, respectively, fungal
mycelia were homogenized in a frozen state. Five flasks were
examined for each treatment. Extraction procedure was as
explained above for plant roots with only minor modifications.
RESULTS
Selection of Fungal Isolates. Three (MAJ, SCO1, and 031)
of the 11 isolates of P. involutus tested were found to
consistently form ectomycorrhiza with poplar, whereas the
others failed (Table 1). Failure to associate successfully may
indicate that the isolates were incompetent with poplar. The
table also showed variations in the growth ability within the
species. All fungal isolates grew on modified MMN agar,
except isolate B12. However, the growth form of mycelia varied
Vol. 14, 2007
POPLAR-PAXILLUS INVOLUTUS PHYSIOLOGICAL INTERACTION 51
Table 1. Origin, host, mycelial radial growth, and ectomycorrhizal
ability with hybrid poplar of isolates of P. involutus (Batsch)
Fr. used in this study
Isolate Origin
Host* Radial growth (mm)** Ectomycorrhiza***
43.00 ± 4.16
B05 Belgium ND
46.83 ± 5.58
B08 Belgium ND
00.00 ± 0.00
B12 Belgium ND
B13 Belgium ND
27.17 ± 1.57
49.50 ± 2.22
+
MAJ France
Poplar
Oak
78.83 ± 2.48
NAU France
Birch/pine
31.00 ± 2.38
P 1 9 Poland
+
85.00 ± 0.00
SCO1 Scotland ND
65.17 ± 3.08
+
031 Germany Spruce
27.83 ± 0.69
032 Germany Spruce
26.17 ± 1.57
533 Germany Spruce
*ND: not determined; **Radial growth was measured three weeks
after the fungi were grown on MMN. Values are expressed as means of
six replicates; ***Ectomycorrhiza was formed (+) or not (-)
Control
among isolates, with particular isolates (SCO1, NAU, and 031)
producing more rapid and dense mycelia during growth on
agar-based media than others. Two isolates with reasonable
growth characteristics, compatible MAJ, and incompetent
NAU (Figure 1), were selected for further experiments. MAJ
was selected for the compatible interaction because the isolate
produced the most consistent ectomycorrhizal association
with poplar.
Morphological and Anatomical Characteristics. Root tips
morphological changes of compatible association
characteristics of mycorrhizal formation were visible one week
after inoculation. The mycorrhiza continued to develop and
they were mature by five to seven weeks. Development of
poplar both grown in the absence and presence of P. involutus
under currently described Petri dish system occurred normally.
Incompetent
Compatible
Figure 1. Root systems of poplar in control plant (left) and plants inoculated with compatible MAJ (middle) and incompetent NAU (right)
P. involutus isolates.
Control
Compatible
5.0 mm
Incompetent
2.5 mm
2.5 mm
R
M
H
0.1 mm
Figure 2. Morphology of poplar root tips (top) and anatomical characteristics of the cross sections of the root tips (bottom) in control,
compatible, and incompeten isolates of P. involutus four weeks after inoculation. H: Hartig net, M: mantle, R: root hair. Control plants
were grown in the absence of the fungus.
52
GAFUR ET AL.
HAYATI J Biosci
600
nkat/g protein
500
400
300
200
100
0
R_COMP
R_NONCOMP
R_CTRL
Figure 3. Peroxidase activities in interaction between poplar and P.
involutus. Data were taken four (left bar) and seven (right
bar) weeks after inoculation. Values are expressed as means
of five replicates. Peroxidase activities were induced only
in incompetent interactions (R_NONCOMP), not in
compatible associations (R_COMP) or in control plants
(R_CTRL).
All the plants exhibited a well-developed root system (Figure
1). Mycorrhiza formed by association between poplar and
compatible isolate of P. involutus were initially pale white to
yellowish and then became brown with age. Mycorrhized
plants in this study were characterized among others by
dramatic changes in root morphology such as stimulation of
lateral root production and suppression of root hair formation.
As shown in Figure 2 (top), root hair formation was restricted
in compatible association. Anatomical characteristics of the
roots of control, compatible, and incompetent associations
were shown in Figure 2 (bottom). The mantle of compatible
isolate surface consisted of loose web-like prosenchymatous
elements. Mantle thickness varied and was irregularly layered.
Hartig net developed showed intercellular penetration by
hyphae, mostly in the form of a single hyphal row, to the first
cortical layer. Upon maturity, the Hartig net fully enveloped
the cells of epidermal layer forming a periepidermal net.
Peroxidase Accumulation. Peroxidase activity increased
significantly in roots of poplar in response to incompetent
isolate of P. involutus (Figure 3). Elevated levels of the
enzymatic activity were maintained seven weeks after
inoculation. In contrast, no significant changes in peroxidase
activity were observed in compatible tissues after inoculation
with mycorrhizal isolates MAJ or in control roots.
DISCUSSION
The results of the present study support the view that
P. involutus has a broad variation of host range at the isolate
level, as this has been previously reported by Heslin and
Douglas (1986) and Molina and Trappe (1982). A number of
studies also attest to significant variation in the ability of
isolates of other fungal species to form ectomycorrhiza with
particular host taxa (Bonfante et al. 1998; Cairney 1999). Similar
growth characteristics of different ectomycorrhizal fungal
species have also been earlier reported by KieliszewskaRokicka (1992) and Littke et al. (1984).
Despite the fact that both growth characteristics and
mycorrhizal ability of P. involutus vary among the tested
isolates, the two traits do not seem to be correlated in the
fungus. For example, isolates NAU and 031 whose growth
rate is at a similar level, differ in their ability to form
ectomycorrhiza. While isolate 031 was able to form
ectomycorrhiza with poplar, isolate NAU failed. In contrast,
the growth rate of compatible isolates MAJ and 031 was
different. Nevertheless, this lack of correlation between the
two traits of mycelial growth and ectomycorrhizal ability
enabled us to select isolates perfectly suitable for further
experiments, i.e. compatible and incompetent isolates with
optimal growth ability.
Increased peroxidase activity has been reported in many
pathogenic interactions between plants and fungi (Svalheim
& Robertsen 1990; Asiegbu et al. 1994; Fossdal et al. 2001).
The fact that overall peroxidase activity was not significantly
increased in the mycorrhizal roots of poplar supports earlier
observations that mycorrhizal colonization does not activate
full-scale plant-defense responses (Nylund 1981; Nylund 1987;
Münzenberger et al. 1997) or elicitation of defense responses
is avoided (Mohr et al. 1998). In the meantime, failure to detect
peroxidase activities to an appreciable amount in short roots
of control plants may well explain that defense-related
peroxidases, in the absence of root microbial pathogens or
other organisms, are not expressed.
As a defense mechanism, plants are able to respond to
invading organisms in a variety of ways including production
of components which modify cell walls including peroxidases,
making them more difficult to penetrate (Hahlbrock et al. 1986).
In symbiotic associations, on the other hand, both cell wall
bound peroxidase and chitinase increased above control levels
during the first days of mycorrhization, only to return to or
fall below those of non-mycorrhizal roots at later stages of
established symbiosis (Salzer & Boller 2001). However,
Bonfante-Fasolo and Spanu (1992) pointed out that most of
the experiments had not always been of a comparative nature,
and a more systematic approach considering both compatible
and incompetent mycorrhizal and pathogenic associations
was needed. The present study, therefore, sheds some light
on and contributes to better understanding of the mechanism
of recognition in plant-microbe interactions.
Accumulation of peroxidase was not induced by all isolates
of P. involutus tested in pure culture (data not shown). All
isolates, including poplar-incompetent, did not seem to induce
peroxidase accumulation to an appreciable amount. Similar
results that no peroxidase activities are detected in extracts of
extramatrical mycelium or pure culture mycelium have also
been reported in different isolates of Suillus bovinus and
P. involutus by Timonen and Sen (1998). Cairney and Burke
(1998) also noted that there is currently no convincing evidence
to support production of peroxidase activity by
ectomycorrhizal fungi in vitro.
Isolates of MAJ and NAU served as perfect models to
investigate physiological interactions between poplar and
P. involutus. At the genomic level we have shown that the
two isolates are very similar with DNA sequence identity of
98.9% although the difference is more significant in terms of
expressed genes after contact with birch roots (Le Quéré et
al. 2004). Thus, the currently described novel system should
POPLAR-PAXILLUS INVOLUTUS PHYSIOLOGICAL INTERACTION 53
Vol. 14, 2007
also enable investigations on ectomycorrhizal interactions in
different systems.
ACKNOWLEDGEMENT
This work was partially supported by a research fellowship
from the Alexander von Humboldt Foundation, Bonn, Germany
to Abdul Gafur. We thank Rosemarie Langenfeld-Heyser,
Christine Kettner, and Gisbert Langer-Kettner for technical
assistance.
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